柱前衍生化HPLC法测定天麻提取物中18种氨基酸含量

Determination of 18 amino acids in Rhizoma gastrodiae extract by precolumn derivation HPLC

目的:建立柱前衍生化高效液相色谱(high performance liquid chromatography,HPLC)法测定天麻提取物中18种氨基酸的含量。方法:供试品在6 mol·L-1盐酸中110℃水解24 h,与异硫氰酸苯酯进行衍生化反应。采用Waters Symmetry^■ C18色谱柱(250 mm×4.6 mm,5μm),流动相A为0.1 mol·L-1乙酸钠缓冲液(pH 6.5)-乙腈(97∶3),流动相B为乙腈-水(80∶20),以1.0 mL·min^-1的流速进行梯度洗脱,检测波长为254 nm,柱温为35℃。结果:18种氨基酸衍生物能够完全分离(分离度>2.0),在1.0~10.0μg·mL^-1浓度范围内线性良好(r>0.999),且供试品溶液在16 h内稳定,平均回收率(n=6)在92.5%~103.8%之间,相对标准偏差(relative standard deviation,RSD)均<3.0%。经检测,3批天麻提取物中氨基酸总含量分别为4.51%,4.20%和4.18%。结论:经方法学验证,本法可适用于天麻提取物中18种氨基酸含量的测定。

Objective: To establish a precolumn derivation HPLC method for the determination of 18 amino acids in Rhizoma gastrodiae extract. Methods: The sample was hydrolyzed in 6 mol·L-1 hydrochloric acid at 110 ℃ for 24 hours,and derivatized with phenyl isothiocyanate( PITC). The test derivatization solution was analyzed by a Waters Symmetry^■ C18 column( 250 mm × 4. 6 mm,5 μm) with a gradient elution in a flow rate of 1. 0 mL·min-1 using mobile phase A( 0. 1 mol·L-1 Sodium acetate buffer of pH 6. 5-acetonitrile = 97 ∶ 3) and mobile phase B( acetonitrile-water = 80∶ 20). The detection wavelength was 254 nm and the column temperature was set at 35 ℃.Results: The 18 amino acids were completely separated with the resolution above 2. 0 in a good linear range of 1. 0 ~10. 0 μg·mL-1( r > 0. 999). The test solution was stable for 16 hours. The recoveries of the 18 amino acids( n =6,RSD < 3. 0%) were between 92. 5% and 103. 8%. The total contents of the amino acids in three batches of Rhizoma gastrodiae extract were 4. 51%,4. 20%,4. 18%. Conclusion: Based on the method validation,this method is suitable for the quantification of the 18 amino acids in Rhizoma gastrodiae extract.